Cyclic pulsation stress promotes bone formation of tissue
engineered laminae through the F-actin/YAP-1/β-Catenin signaling
axis

Ethics statement

We have complied with all relevant ethical regulations for animal testing and research. All animal experiments were performed in the Animal Facility of East China Normal University and according to the protocol (Protocol number: 20150482A168) authorized by the Animal Care and Use Committee of Fudan University.

Cell culture and transfection

Rat mesenchymal stromal cell derived from Wharton’s Jelly were obtained from the Otwo Biotech (Guangzhou, China). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, UT, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Beit HaEmek, Israel).

Adenoviral constructs containing full-length β-CATENIN (Han-Bio, Shanghai, China) were used to generate stable β-CATENIN overexpressing cell lines. In all, 1 × 105 MSC cells were plated in a 24-well plate and infected with β-CATENIN adenovirus at a multiplicity of infection of 100 in the presence of Lipofectamine 2000 (Invitrogen, CA, USA, 2 μl). After 24 h of incubation with adenovirus, cells were harvested for the determination of mRNA or protein expression levels of β-CATENIN.

For the establishment of transient β-CATENIN-knockdown or YAP1-knockdown cells, MSC was transfected with siRNAs (Ribo-Bio, Guangzhou, China). The sequences of siRNAs were listed as follows: si-β-CATENIN, 5′-GCACCATGCAGAATACAAA-3′; si-YAP1, 5′-GGTCAGAGATACTTCTTAA-3′. In all, 1 × 105 MSC cells were plated in a 24-well plate and infected with siRNAs (50 nM) in the presence of Lipofectamine 2000 (2 μl). After 24 h of incubation, cells were harvested for the determination of mRNA or protein expression levels of β-CATENIN or YAP1.

Inhibitors or agonists preparation

PD98059, XAV-939, wortmannin, 6-bromoindirubin-3′-oxime (BIO), and Cytochalasin B were purchased from MedChemExpress (NJ, USA). The chemical reagents were dissolved in dimethyl sulfoxide at a concentration of 10 mM, 10 mM, 1 mM, 10 mM, and 10 mM, respectively. And the final concentration of the chemical reagents in the culture medium was: 20 μM, 1 μM, 25 nM, 2 μM, and 20 μM, respectively.

Bioreactor setting

To construct an in vitro cyclic pulsation mechanical model mimicking rat lumbar CSFP, the bioreactor setting parameters were set as below: flow rate at 15 cm/s, solenoid valve frequency at 300 times/min. MSC at passage 4–7 was evenly pipetted on sterile [poly(lactic-co-glycolic acid), PLGA] membranes (Nuoqi, Chongqing, China) to construct MSC-PLGA complexes. The MSC-PLGA complexes were divided into the cerebrospinal fluid pulsation (CSFP) group and cerebrospinal fluid pulsation isolation (Non-CSFP) group. In the CSFP group, the MSC-PLGA complexes were fixed onto the pulsation tube by the clip and connected to the bioreactor; while in the Non-CSFP group, the MSC-PLGA complexes were also fixed onto the pulsation tube by the clip and not connected to the bioreactor.

Reverse-transcription PCR

Total RNA was isolated from cells or bone tissue using TRIzol Reagent (Invitrogen) according to manufacturer’s instructions. cDNA was synthesized from total RNA (500 ng) using reverse transcription kit (Takara, Tokyo, Japan). qPCR was performed in triplicate using 1 µl of cDNA in a standard SYBR premix Ex Taq (Takara) on the Applied Biosystems 7500 Real-Time PCR Detection System (Applied Biosystems, CA, USA). GAPDH served as an internal control. The following primers were used: GAPDH, 5′-GGCACAGTCAAGGCTGAGAATG-3′, 5′-ATGGTGGTGAAGACGCCAGTA-3′; C-MYC, 5′-GGTGGAAAACCCGACAGTCA-3′, 5′-TAGCGACCGCAACATAGGAC-3′; CYCLIN-D1, 5′-ACCAATCTCCTCAACGACCG-3′, 5′-CTCCTCGCAGACCTCTAGCA-3′; β-CATENIN, 5′-CCAAGTGGGTGGCATAGAGG-3′, 5′-CAGGCTCGGTAATGTCCTCC-3′; YAP1, 5′-CTTGACCCTCGTTTTGCCATGAA-3′, 5′-GACGGTCTGACATTTTGGAGCAT-3′; RUNX2, 5′-ATGGTTAATCTCTGCAGGTCACT-3′, 5′-CTGCTTGCAGCCTTAAATGA-3′; OSTERIX, 5′-TGAGGAAGAAGCCCATTCAC-3′, 5′-ACTTCTCCCGGGTGTG-3′; BMP2, 5′-CAGCGAGTTTGAGTTGAGG-3′, 5′-CGGTACAGGTCGAGCATAT-3′; OCN, 5′-CCTAGCAGACACCATGAGGA-3′, 5′-GTCAGAGAGGCAGAATGCAG-3′; ALP, 5′-GCACAACATCAAGGACATCG-3′, 5′-TCAGTTCTGTTCTTGGGGTACA-3′; VEGF-A, 5′-CGAACGTACTTGCAGATGTGAC-3′, 5′-GACCCAAAGTGCTCCTCGAA-3′; FGF2, 5′-GCGACCCACACGTCAAACTA-3′, 5′-ACTGGAGTATTTCCGTGACCG-3′; MMP9, 5′-ACGTCTTTCACTACCAAGACAAG-3′, 5′-GCAGGAGGTCATAGGTCACG-3′.

EdU assay

Cell proliferation was measured with an EdU assay kit (Ribo-Bio, Guangzhou, China). Experiments were performed according to the manufacturer’s instructions. The representative images were obtained using a Leica fluorescence microscope.

Red phalloidin staining

Cytoskeleton of MSC was stained with Acti-stain 555 Fluorescent Phalloidin (Cytoskeleton, CO, USA, 1:500) according to the manufacturer’s instructions.

Immunofluorescence

Immunofluorescence was performed as previously described10. The following antibodies were used: β-Catenin mouse monoclonal (1:100), YAP1 mouse monoclonal (1:100), Runx2 mouse monoclonal (1:200), Osterix mouse monoclonal (1:500), VEGF-A mouse monoclonal (1:100), mouse IgG1-FITC isotype control (1:200, Santa Cruz, CA, USA), β-Catenin rabbit polyclonal (1:100), CD31 rabbit monoclonal (1:500, Severicebio, Wuhan, China), Rabbit IgG monoclonal isotype control (1:200, Abcam, MA, USA), Alexa Fluor® 647 conjugated Goat Anti-Mouse IgG (1:100), Alexa Fluor® 488 conjugated Goat Anti-Mouse IgG (1:100, Abcam, MA, USA), Cy5 conjugated Goat Anti-rabbit IgG (1:100), Alexa Fluor® 488-conjugated Goat Anti-Rabbit IgG (1:100), Cy3 conjugated Goat Anti-Rabbit IgG (1:100, Severicebio), and DAPI (Leagene, Beijing, China).

Immunohistochemistry

Immunohistochemistry was performed as previously described10. The following antibodies were used: BMP2 rabbit polyclonal (1:500), OCN rabbit polyclonal (1:200), VEGF-A rabbit monoclonal (1:300), and horseradish peroxidase (HRP)-conjugated secondary antibodies (Severicebio), mouse IgG1-FITC isotype control (1:200, Santa Cruz), Rabbit IgG monoclonal isotype control (1:200, Abcam).

Co-immunoprecipitation

Cells were lysed in IP buffer (Servicebio) containing protease inhibitor cocktail (Servicebio). Total lysates (200 μg) were incubated with primary antibodies (1 µg) and Protein A/G agarose beads (Millipore, MA, USA) overnight at 4 °C with gentle shaking. The immunoprecipitated complexes were then washed with lysis buffer three times and eluted from the beads with protein loading buffer. The immunoprecipitated complexes were immunoblotted and subjected to mass spectrometry analysis (ClinX, ChemiScope 6300, MA, USA). Images were analyzed using ImageJ software.

Western blotting analysis

Western blot was performed as previously described35. Total protein was extracted from the lysed samples in RIPA buffer with PMSF (Beyotime, Shanghai, China) on ice. Then cells were collected and centrifuged for 15 min (12,000 rpm, 4 °C). BCA Protein Assay kit was used to quantify protein concentration according to the manufacturer’s instructions (Thermo, MA, USA). The supernatants (20 μg protein) were subjected to SDS-PAGE gel and transferred onto PVDF membranes (Millipore). The nonspecific sites were blocked with 5% non-fat dried milk for 2 h at room temperature. The membranes were then incubated with primary antibodies for β-Catenin (Santa Cruz, 1:1000), YAP1 (Santa Cruz, 1:1000), and β-Actin (Santa Cruz, 1:1000) at 4 °C overnight. The membranes were incubated with goat anti-mouse IgG-HRP secondary antibody (Santa Cruz) at room temperature for 2 h. Finally, the ECL Detection kit (Beyotime, Shanghai, China) and Fluor Chem E system (Proteinsinple, CA, USA) were used for detection and photography. Images were analyzed by ImageJ software. All blots were derived from the same experiment and processed in parallel. Uncropped western blotting images were provided in Supplementary Fig. 4, where the size markers were labeled.

Construction of tissue-engineered laminae

The hydroxyapatite-collagen I scaffold was bought from the Beijing Allgens Medical Science & Technology Co., Ltd. Under the sterile condition, the hydroxyapatite-collagen I scaffold was cut to the size of 8 mm × 6 mm. Rat MSC at passage 4–7 was trypsinized and resuspended in media at a concentration of 1 × 106/ml. Then, 100 μl cell suspensions were pipetted on one side of each scaffold, and after 30 min, 100 μl cell suspensions were pipetted on the other side. All constructs were placed in culture medium before implantation.

Live/Dead staining

Cell viability of MSC in TEL was measured with a Live/Dead assay kit (Best-Bio, Shanghai, China). Experiments were performed according to the manufacturer’s instructions. The representative images were obtained using a ZEISS confocal fluorescence microscope (ZEISS, Jena, Germany).

Scanning electron microscope

The TEL were fixed with electron microscopy fixative solution (Severicebio), dehydrated, mounted on an aluminum stub, and sputter-coated with gold-palladium for 30 s. The morphology of TEL and MSC adhesion on the scaffolds was then viewed on a scanning electron microscope (Hitachi, Tokyo, Japan).

Animal studies

The construction of CSFP and Non-CSFP were performed as previously described8,9,10. Briefly, for the CSFP animal model, spinous processes and interspinous ligaments were removed to expose the vertebral laminae, a bone defect measuring 8 mm × 6 mm × 1 mm was created in the vertebral laminae, leaving two fresh cancellous bone end measuring 8 mm × 1 mm × 2 mm, then the TEL were placed and fixed in the bone defect. For the Non-CSFP animal model, spinous processes and interspinous ligaments were removed to expose the vertebral laminae, a cancellous bone end measuring 8 mm × 2 mm was created in the outer cortex of laminae while preserving the dura surface cortex of laminae, similar to that of the CSFP group, then the TEL were fixed onto the native laminae. For the CSFP + XAV animal model, the surgical procedure was the same as the CSFP animal model; but after the procedure, the rats were injected intraperitoneally with XAV-939 according to the standard of 4 mg/kg at the frequency of twice a week for the first two weeks and once a week. (Supplementary Fig. 2).

Statistical analysis

Statistical analysis was conducted using GraphPad Prism version 6.02 software program for Windows (GraphPad, CA, USA). All data are presented as mean ± SEM. One-way ANOVA or two-way ANOVA were used for analysis, and Tukey’s multiple comparisons test was used for comparison between groups. All tests were two-sided, and P-values < 0.05 were considered to be statistically significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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